Work on the role of insulin-like growth factors (IGFs) in pediatric sarcomas is continuing to focus on potential therapeutic approaches as well as further defining the downstream signaling events of IGF action. We have continued to evaluate several humanized monoclonal antibodies directed against the IGFIR as well as a small molecule kinase inhibitor with specificity against the IGFIR kinase. We have chosen to focus on the antibody as this has proven to have better activity in our models, and we are evaluating this in the clinic as well. We also have continued to analyze the potential interactions between IGFIR blockade and mTOR inhibition. We have now clearly documented activity of the IGFIR Ab in human rhabdomyosarcoma (RMS) xenografts and shown that the activity appears to correlate well with the level of IGFIR receptor expression, with those xenografts expressing high levels of IGFIR being the most sensitive to the antibody. Response in these xenograft models also is correlated with a rapid decrease in AKT phosphorylation. However, we have now found that by day 60, the levels of AKT phosphorylation are back to baseline, and this is associated with a demonstrable escape and regrowth of the tumors. We believe these data suggest the need for combination therapy, and are currently attempting to determine what pathways are altered as the xenografts escape from the antibody-induced growth inhibition. We have shown that combining the antibody treatment with rapamycin leads to a more profound tumor-growth inhibition. Furthermore, rapamycin treatment alone led to rapid phosphorylation of AKT while combining this with the IGFIR antibody abrogated that unwanted effect. These studies have provided a strong rationale for considering the combination of IGFIR blockade along with mTOR inhibition in the clinical setting, and we hope to follow our clinical study of the IGFIR antibody with just this combination in the clinic. The rationale for mTOR inhibition of these tumors as a therapeutic approach has been strengthened by our recent analysis of primary RMS tumors using reverse-phase proteomics demonstrating that activation of the mTOR signaling pathway in these tumors is a poor prognostic factor. We have continued to study the molecular mechanisms of ezrin signaling in pediatric sarcomas. We previously linked ezrin to mTOR signaling, and current work has suggested that beta-4 integrin signaling also is required for metastatic functioning in a human osteosarcoma xenograft model. We currently are working to determine whether there is a link between ezrin and beta-4 integrin in the mestastatic behavior of these cells. We completed our work with CXCR-4 chemokine signaling in osteosarcoma, and although we could show an effect on metastatic behavior, we have elected to not pursue this clinically due to several concerns. We have instead focused our attention on creating an in vitro model of tumor cell-stromal cell interactions in osteosarcoma, so that we may begin to dissect out critical interactions between tumors cells and their environment that may identify novel therapeutic targets. To date, we have created a culture system involving human osteosarcoma cells, fibroblasts and a nanofiber lattice and are working out conditions to harvest individual cell components to study protein and RNA expression alone and in the co-culture conditions. Ultimately, we hope to identify up-regulated pathways due to cell-cell interactions.